Measuring Action Potential Propagation Velocity in Murine Cortical Axons

Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of propagation velocity using a combination of somatic whole cell and axonal loose patch recordings in brain slice preparations. The axons of neurons filled with fluorescent dye via somatic whole-cell pipette can be targeted under direct optical control using the fluorophore-filled pipette. The propagation delays between the soma and 5–7 axonal locations can be obtained by analyzing the ensemble averages of 500–600 sweeps of somatic APs aligned at times of maximal rate-of-rise (dV/dtmax) and axonal action currents from these locations. By plotting the propagation delays against the distance, the location of the AP initiation zone becomes evident as the site exhibiting the greatest delay relative to the soma. Performing linear fitting of the delays obtained from sites both proximal and distal from the trigger zone allows the determination of the velocities of AP backward and forward propagation, respectively. Key features • Ultra-thin axons in cortical slices are targeted under direct optical control using the SBFI-filled pipette. • Dual somatic whole cell and axonal loose patch recordings from 5–7 axonal locations. • Ensemble averaging of 500–600 sweeps of somatic APs and axonal action currents. • Plotting the propagation delays against the distance enables the determination of the trigger zone's position and velocities of AP backward and forward propagation.

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.81463 Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation.This protocol describes the measurement of propagation velocity using a combination of somatic whole cell and axonal loose patch recordings in brain slice preparations.The axons of neurons filled with fluorescent dye via somatic whole-cell pipette can be targeted under direct optical control using the fluorophore-filled pipette.The propagation delays between the soma and 5-7 axonal locations can be obtained by analyzing the ensemble averages of 500-600 sweeps of somatic APs aligned at times of maximal rate-of-rise (dV/dtmax) and axonal action currents from these locations.By plotting the propagation delays against the distance, the location of the AP initiation zone becomes evident as the site exhibiting the greatest delay relative to the soma.Performing linear fitting of the delays obtained from sites both proximal and distal from the trigger zone allows the determination of the velocities of AP backward and forward propagation, respectively.f.Take the black buffer tray and specimen holder from the freezer and place the black buffer tray on ice.g.Wipe the specimen holder.Note: The holder should be completely dry.h.Place a small amount of instant tissue adhesive on the center of the specimen holder.i. Fill the Petri dish with cold slicing solution (step A1).p. Wait a minute for the isoflurane to take effect.q.Take a mouse and cut the skin from its head to expose the skull.r.Cut the head close to occipital bone with scissors.s.Wash the head in the first weighing dish with cold slicing solution for a few seconds.t.Cut the bone with surgical scissors as follows:

Materials
i. Insert the surgical scissors through the foramen magnum in the occipital bone.
ii. Continue along the occipital bone until the end of the lacrimal bone.
iii.Cut down from the lacrimal's end to the eye cavity.u.Gently remove the cut bone with tweezers and expose the brain.v. Put the brain into the second weighing dish with ice-cold slicing solution.w.Transfer the brain into a Petri dish.
x. Cut the block of the brain (coronal or sagittal plane).y.Paste it in the specimen holder.z.Transfer the holder to a buffer tray.aa.Insert the buffer tray into the insert in the vibratome.bb.Fill the buffer tray with an ice-cold slicing solution.cc.Cut the first thick slice.dd.Discard the slice.ee.Adjust to 300 µm slice thickness and cut the slice.ff.Remove the slice with a paintbrush.gg.Collect the slice with a cut Pasteur pipette.hh.Transfer the slice to a warm incubation solution (step A2).ii.Repeat until the desired number of slices is cut.jj.Incubate the slices for 60 min.Caution: It is advisable to perform steps 4r-4ee with maximum speed, ideally completing them in under 1 min.More extended periods of ischemia/hypoxia could cause irreversible damage to the brain tissue.

B. Whole-cell recording and filling neurons with fluorescent dye (always document the experiment in a lab book)
Published: Nov 05, 2023 11. Apply gentle positive pressure, place the pipettes in the bath, focus on their tips, and move them down, one by one, toward the surface of the slice.12. Stop moving the pipettes before approaching the cell; ensure that the cell body is below the whole-cell pipette.

C. Measuring action currents along the axon
1.After establishing the Current Clamp whole-cell recording with pipette 1, wait 15 min to allow SBFI diffusion to the cell.2. Focus the NeuroCCD-SMQ camera on the axon.L5 cell axons are the only fine processes emerging from the soma opposite the apical dendrite and exhibiting distinctive Na + transients (Fleidervish et al., 2010; Baranauskas et al., 2013).Some neurons were examined live using a two-photon microscope after the physiological experiment.In these cells, axons were easily discerned from basal dendrites due to their lack of spines, confirming earlier observations during recording.3. Acquire an 8 ms long single frame image with a NeuroCCD-SMQ camera using the NeuroPlex software.
Use 385 nm high-intensity LED device as a light source and collect emission using the U-MNU2 filter set.The 8 ms long exposure time is sufficient to obtain a high-quality image while minimizing light intensity that might induce photo-dynamic damage to the cell.4. Find a point of interest along the axon.The first point of interest could be at ~10 µm from the edge of the soma if the backpropagation velocity has to be measured; in experiments designed to measure the forward propagation, the first point of interest might be more distal than the presumed trigger zone, ~30-40 µm from the edge of the soma. 5. Bring pipette 2 to the point of interest (Figure 1b).6. Touch the axon with the pipette 2. Ensure the touch by taking one or more single frames with the NeuroCCD-SMQ camera.The proper touch could be established by fine-tuning the pipette 2 position, with no suction applied to the pipette.7. Deliver 2-7 ms long current steps via pipette 1. Increase the current step amplitude until it will be sufficient to elicit an AP. 8. Deliver 600 steps of 1.5× threshold amplitude at 2-5 Hz frequency.Record the voltage with pipette 1 and current with pipette 2. 9. Store the recording file for further analysis.10.Find another point of interest (Figure 1c).It should be 10-20 µm further distally from the previous point of interest to ensure a significant time delay difference.11.Repeat steps 5-9 for 6-10 points of interest within 10-150 µm of the axonal length.

4 Published
Titrate to pH 7.25 with KOH 1 M Due to the large size and spatial complexity of L5 pyramidal neurons in slices, ATP and GTP supplements were not added to the intracellular solution.The freshly prepared K + -based intracellular solution was tested in wholecell recording from several neurons before supplementing with SBFI.The SBFI-containing solution was separated in aliquots of 100 µL into small Eppendorf microtubes and stored at -20 °C.EquipmentPublished: Nov 05, 2023ProcedureA.Preparation of brain slicesCoronal or sagittal slices are suitable, as these plains of section preserve the axonal and apical dendritic tree of L5 pyramidal neurons.1. Preparing the solution for brain exposure and slicing: a. Transfer 250 mL of freshly prepared aCSFx1 solution into a 300 mL chemical glass beaker.b.Place the glass beaker into the ice box.c.Bubble the aCSFx1 solution with 95% O2-5% CO2 gas mixture for at least 15 min to attain equilibrium in gas partial pressures.d.Cover the glass beaker with Parafilm.e. Cool the solution to 4 °C.f.Add 0.7 mL of MgSO4 1 M. g.Add 0.25 mL of CaCl2 1 M (prior bubbling with 95% O2-5% CO2 gas mixture is necessary to prevent Ca 2+ ions precipitation).h.Cover the chemical glass with Parafilm.2. Preparing the incubation solution for brain slices: a. Transfer 500 mL of freshly prepared aCSFx1 solution into the slice incubator.b.Bubble with 95% O2-5% CO2 for at least 15 min.c.Cover the slice incubator with Parafilm.d.Warm the aCSFx1 solution to 30 °C.e. Add 0.7 mL of CaCl2 1 M. 3. Preparing the vibratome: a.Put the black buffer tray and specimen holder into the freezer (-20 °C).b.Take the double-edge feather blade, wash it with 70% ethanol, rinse with water, and wipe.c.Insert the blade into the blade holder.d.Insert the Vibrocheck unit into its slot and connect to the vibratome by the USB cable.e. Wait for the appearance of a green light on the Down button.f.Insert the Allen screwdriver into the blade holder slot and rotate it 90° counterclockwise until a white strip appears on the left side.g.Push the Down button.h.Push the Run button.i.If the display shows a value different from 0 (e.g., -0.1), push the Stop button.j.Release the blade.k.Insert the screwdriver in the adjustment hole opening and turn in the "-" direction.l.Push the Run button.m.Repeat if the display shows a value different from 0. n.After reaching 0, push the "Down" button to remember the parameters.o.Move the blade to the top position and remove the Vibrocheck block.4. Slicing procedure (done in the chemical hood): a.Put the Whatman filter paper 90 mm into the 100 mm Petri dish and place the dish on ice.b.Prepare the single edge, carbon steel blade.c.Prepare surgical instruments: scissors, small mini-scissors, tweezers, spatula, and Pasteur cut pipette.d.Bring the mouse from the animal facility.e. Check the temperature of the solutions.

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Cite as: Kotler, O. et al. (2023).Measuring Action Potential Propagation Velocity in Murine Cortical Axons.Bio-protocol 13(21): e4876.DOI: 10.21769/BioProtoc.4876.Nov 05, 2023 j.Put two weighing dishes near the surgical instruments.k.Fill two weighing dishes with cold slicing solution.l.The next steps have to be performed in accordance with the Institutional and National Animal Care regulations.m.Measure 0.5 mL of isoflurane and distribute it over the towel attached to the cover of the exicator.n.Cover the exicator.o.Put the animal into the exicator and quickly cover it.

13 . 14 . 16 . 18 .
Set the Multiclamp-700B Commander Channels 1 and 2 to voltage-clamp mode and correct the pipette offset.Apply a seal test, a -10 mV voltage step, to both pipettes.15.Approach the cell body with the first pipette by moving it along the z-axis until a dimple appears on the membrane.Release the positive pressure and apply gentle suction while monitoring the current response to the voltage step until the gigaohmic seal forms.17.Move the holding voltage to -70 mV and correct fast capacitance using automatic correction of the Commander.Apply gentle and brief suction pulse with a syringe to break into whole-cell configuration (or by mouth).The successful break-in is evident by an abrupt increase in the amplitude of capacitative transients in response to -10 mV voltage steps.19.Switch the Commander to the Current Clamp mode.20.Correct the Bridge.21.A Current Clamp whole-cell recording is established for pipette 1.

. Artificial cerebrospinal fluid solution (aCSF)-working solution Reagent Final concentration Amount for 1 L
Bubbling with 95% O2-5% CO2 gas mixture is necessary to avoid precipitation of the divalent ions.